Separation of biomolecules is commonly achieved by means of affinity reaction employing the specific binding of a biomolecule with its binding partner immobilized on a solid support. Bioaffinity separation is defined as an affinity separation in which at least one of the components involved in the affinity reaction is biologically active or is of biological interest. Bioaffinity separations generally involve at least one biomacromolecule such as a protein or nucleic acid, as one of the components of the binding pair. Examples of such binding pairs include: antigen-antibody, substrate-enzyme, activator-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding proteins-vitamin, binding protein-nucleic acid, reactive dye-protein and others. The terms ligand and binder for the ligand will be used to represent the two components of a specific binding pair, the binder being complementary to the ligand of interest.
The most common means used to effect the separation of the bound material from that remaining unbound is to attach either a ligand or binder for the ligand to a solid support; then once the specific binding has occurred, to remove the solid from the liquid environment. This attachment to a solid support can be either covalent or noncovalent and can be either directly to the support or via a linker between the support and ligand or binder for the ligand.
Affinity separation is commonly a step in many multi-step processes. One example is the area of heterogeneous immunoassays. Here a bioaffinity separation is used to capture an analyte from a complex mixture, frequently, serum or plasma. After capturing the analyte, the contaminants are washed away and the analyte detected using any number of well-known assay protocols. Some solid supports used in heterogeneous immunoassays are 1/4 inch plastic spheres, the inside of test tubes, the inside of microtiter plate wells, latex particles and magnetic particles.
Microparticles such as latex particles or magnetic particles are often preferred solid supports for use in affinity separation and heterogeneous immunoassay. Microparticles offer a high surface area providing enhanced coupling capacity for a ligand or binder for the ligand. In addition, microparticles remain substantially dispersed during a specific binding reaction allowing the immobilized ligand or binder for the ligand to be intimately mixed with the target binder for the ligand or ligand in the sample which shortens the reaction time. Since microparticles are substantially suspendible in solution, the slurry can be treated as a liquid reagent and delivered reproducibly and quantitatively which is an added advantage for easy handling of the solid support. However, separation of these particles is often tedious and labor intensive requiring centrifugation, filtration or a special magnetic separation device.
Despite the high magnetic field gradient requirement for separation, magnetic particles have been widely applied to heterogeneous immunoassays due to high surface area, efficiency of separation and ease of handling. Various types of magnetic particles suitable for use in immunoassay have been described by, for example, Hersh, et al., U.S. Pat. No. 3,933,997; Ithakissios, U.S. Pat. No. 4,115,534; Forrest, et al., U.S. Pat. No. 4,141,687; Mansfield, et al., U.S. Pat. No. 4,197,337; and Chagnon, et al., Danish Application DK 2,374,184.
The concept of using microparticles, particularly magnetic particles, to effect separation of bioactive materials as in immunoassays, has been extended over time to include affinity purification of enzyme, proteins or micro-organisms applicable to any adsorption-desorption process [Dunhill, P. et al., Biotech. Bioeng. (1974) 10, 987-990; Horisberger, M., Biotech. Bioeng. (1976) 18, 1647-1651]. In these applications, a simple and fast separation technique for the microparticles is also essential.
A flocculating agent is a substance which facilitates and accelerates aggregation of particulate matters from a suspension ultimately leading to precipitation of the aggregates in some situations. Flocculating agents are most commonly used in water purification for removal of both organic and inorganic particulate matters. Traditional flocculating agents include potassium sulfate, ferric chloride and ferric sulfate. More recently, polymeric flocculating agents have also been used for water purification applications. There are many other agents such as polyethyleneimine (PEI) or PRIMAFLOC.RTM. flocculating reagent (Rohm and Hass, Philadelphia, Pa.) known in the art.
Hou, et al., U.S. Pat. No. 4,578,150, describes a filter device containing silica particles for the purposes of removing certain biological materials from a sample by adsorption. The silica particles are added simply to enhance the filtration efficiency. And a flocculating agent is added to improve the retention of the silica particles in the filter.
Chromatographic supports containing immobilized PEI have been described by Alpert, et al. in J. of Chromatography, Volume 185, 375-392, 1979; Vanecek, et al. in Analytical Chemistry, Volume 121, 156-159, 1982; and Flasher in EP Application 0,162,462. In all of these applications, PEI is used to provide ion-exchange properties for purification of proteins, particularly IgM class monoclonal antibodies, not as a flocculating agent.
While it is highly desirable to employ microparticles as solid support in affinity separation or heterogeneous immunoassay, inconvenient separation techniques of such particles have limited the utility, especially use on automated instruments. The method of this invention provides a simple, rapid and inexpensive means of separating microparticles using flocculating agent immobilized on chromatographic support.